1. Field of the Invention
Methods and compositions for the inhibition of growth of infectious Aspergillus fumigatus and other pathogenic mycotic organisms in the gastrointestinal tract of mammalian and avian species.
2. Background
Aspergillosis and mycotic infections. Aspergillosis is an acute systemic mycotic infection caused by Aspergillus sp. Species of Aspergillus known to cause an infection in mammals and avian species include Aspergillus fumigatus, Aspergillus flavis and Aspergillus niger. Infectious aspergillosis has been involved in mycotic abortions and related diseases (Jensen et al., 1991; McCausland et al, 1987), Medical literature contains numerous references indicating an increasing incidence of small bowel infarctions and coagulopathies in humans related to aspergillosis (Catalano et al., 1997; Prescott et al., 1992; Oshawa, 1991). Aspergillus fumigatus has also been implicated as a possible etiological agent in Jejunal Hemorrhage Syndrome in cattle, a new emerging disease that causes massive hemorrhaging of the small intestine (Puntenney et al., 2003). This disease also affects immunocompromised humans Aspergillosis is also documented to cause a high incidence of abortions and pneumonia in cattle, a source of significant economic loss to the livestock industry (Puntenney et al., 2003).
Inoculation of an animal with Aspergillus sp, is commonly through ingestion of mold-contaminated feedstuffs due to improper storage or harvesting techniques, fecal contamination of feedstuffs from birds and rodents, and inhalation of Aspergillus spores from bedding material. The organism is especially effective in evasion of host animal defenses, by secretion of various lipid compounds, including fumigillin, fumitremorgin A, fumigaclavine, and gliotoxin at the infection site, impairing localized generation of complement factors C3a and C5a, thereby blocking recruitment of polymorphonuclear cells. Phagocytic cells normally follow the chemotactic gradient of complement components to the site of the infection, where engulfment and elimination of the organism from the animal occurs (Rhodes et al., 1992). The ability to sequester iron is a virulence factor for microorganisms. The binding of circulating iron to transferrin and lactoferrin, as well as the intercellular storage of iron, reduce levels of free iron below that required for microbial growth. Aspergillus sp, produces two major siderophores (high affinity iron binding compounds) that compete successfully with transferrin and lactoferrin to acquire iron for growth: N,N′,N″-triacetylfusarinine C and ferricrocin. Proteolytic digestion of transferrin may also be utilized as a means of iron acquisition. Iron is released from transferrin at pH<6 as a result of protonation of the iron binding site generally localized at infection site (Rhodes et al., 1992).
Aspergillus sp. produces two serine proteinases, elastinolytic and azocollytic enzymes, which break down tissue (Tomee and Kauffman, 2000; Frosco et al., 1989). Proteinase enzyme production is tied to tissue invasiveness and facilitates entry from colonization site into the parenchyma. Hemorrhagic infarction (vascular blockage) and tissue necrosis may follow infection (Rhodes et al., 1992). After tissue invasion, dissemination via the bloodstream to other organs and the placenta occurs. Placentitis and subsequent abortions commonly result.
The fungal cell wall is composed of highly-branched β-1,3glucans with linkages to chitin, galactomannan and a linear β-1,3/1,4 glucan (Fontaine, et al., 2000). Understanding of the structural biology of pathogenic fungi has aided the design of the invention. The efficacy of the invention is partly related to the addition of β-1,3(4)-endoglucanohydrolase, which delays initiation of fungal growth via enzymatic degradation of cell wall components.
A second component of the invention, which functions in tandem with β-1,3(4)-endoglucanohydrolase, is β-1,3 glucan. The role of β-1,3 glucan in macrophage activation and response to pathogens is well documented in the literature (Czop, et al., 1985, Xia et al., 1999). Upon entering the aqueous conditions of the digestive tract, the large glucan molecules in the invention, via the enzymatic activity of β-1,3(4)-endoglucanohydrolase, are effectively reduced to smaller moieties that may be accepted by macrophage receptors, stimulating immunological activation. Other components of the invention, diatomaceous earth and aluminosilicate, are capable of binding toxic lipid compounds which are secreted by pathogenic fungi. These toxins, if not bound, effectively block release of complement factors required for macrophage response to colonization.
The feeding of a combination of β-1,3(4)-endoglucanohydrolase, β-glucan, calcined diatomaceous earth (hereafter identified as diatomaceous earth, SiO2), glucomannan, and mineral clay, such as aluminum silicate, montmorillonite clay, bentonite or zeolite, has been shown to delay growth of A. fumigatus for a period of several hours in laboratory cultures and clinical trials thereby effectively minimizing the ability of Aspergillus to inhibit complement factor production thereby allowing an intact immune system to respond to fungal colonization.